DE3852918T2 - Pig TGEV virus as a vaccine for dogs. - Google Patents

Abstract

Sterile pharmaceutical compositions for use in therapy especially inactivated vaccines for use in prophylaxis, are prepared by inactivating a live virus or bacterium with ascorbic acid and/or a salt thereof in the presence of oxygen and a source of heavy metal ions. Methods of treating animals, including humans, with such compositions are also disclosed.

Description

This invention relates to vaccines and, in particular, to vaccines that can be used to protect dogs against canine coronavirus.

The coronaviruses were first recognized as a group and morphologically defined by Tyrrell and coworkers. A comprehensive review of the biology and pathogenesis of coronaviruses was published by H. Wege et al., Current Topics in Microbial Immunology,1982,99,165-200.

Canine coronavirus (CCV) was first isolated in 1971 and has become an increasing problem, particularly among young dogs and dogs raised in kennels. It causes mild to moderate diarrhea, often preceded by lethargy, depression, and loss of appetite. Dehydration and subsequent weight loss generally follow. Although the disease is not generally fatal, few puppies are thought to regain full vitality following infection.

Treatment that can be used to overcome the effects of CCV infection involves the administration of massive doses of fluids to replace those lost and, in addition, antibiotics to combat any opportunistic bacterial infection that may strike the dog in its weakened state. Such treatment is expensive and has the disadvantage that it is not preventive and only helps to treat the infection after it has broken out.

A live vaccine introduced by Fort Dodge Laboratories, Iowa, in 1983 to protect dogs against CCV soon proved to be unsafe and was voluntarily withdrawn from the market in the same year (see M.L. Martin in The Compendium on Continuing Education, 1985, 12,1012-1017).

An inactivated vaccine to protect dogs against CCV has now been introduced by Fort Dodge Laboratories under the trade name Duramune Cv-K. The U.S. patents relevant to this product are U.S. Patent No. 4,567,042 and U.S. Patent No. 4,567,043.

Given the infectious nature of CCV and the economic aspects of the disease, new and improved vaccines are still urgently needed.

It is known that canine coronavirus (CCV), transmissible porcine gastroenteritis virus (TGEV), feline infectious peritonitis virus (FIPV) and human respiratory coronavirus of group 229E form an antigenic cluster of viruses within the family Coronaviridae (see N.C. Pedersen et al, Arch. Virol., 1978, 58, 45-53 and S. Siddell et al, J. Gen. Virol, 1983, 64, 761-76).

However, studies of protection by cross-reaction with these viruses have had little or no success. For example, in one study, pigs administered virulent CCV did not produce neutralizing antibodies against TGEV (L.N. Binn et al., Proceedings of the Annual Meeting U.S. Animal Health Association, 1974, 78, 359-366), while in another study, cats inoculated with a virulent field isolate of CCV were not protected against a reactive dose of FIPV (J.E. Barlough et al., Laboratory Animal Science, 1984, 34, 592-597).

Another study has shown that cats vaccinated with virulent TGEV and then challenged with a responding dose of FIPV were not protected against FIPV infection (see, for example, D.J. Reynolds and D.J. Garwes, Arch. Virol., 1979, 60, 161-166; and R.D. Woods and N.C. Pedersen, Vet. Microbiol.,1979, 4, 11-16).

Surprisingly, it has been found that vaccines based on TGEV protect dogs against CCV infection.

Vaccines based on inactivated TGEV are known per se and are described, for example, in GB-A-1 058 340. Their use to prevent CCV infection in dogs has not been previously proposed.

Accordingly, the present invention provides the use of porcine transmissible gastroenteritis virus for the manufacture of a vaccine for preventing canine coronavirus infection in dogs.

The TGEV vaccine can be live or inactivated.

The invention also provides a method for preparing a vaccine composition for preventing canine coronavirus infection in dogs, which comprises mixing a transmissible porcine gastroenteritis virus with a pharmaceutically acceptable carrier.

The TGEV vaccine for use in the invention can be prepared from an isolate of TGE virus, for example the attenuated, cell culture-adapted TGE virus known as Perdue strain TGE virus, which has been deposited with the American Type Culture Collection and has the ATCC accession number VR-763.

Live attenuated TGE viruses suitable for use in the invention can be prepared by passaging the TGE virus isolate in pig and/or cat cells several times, for example 2 to 9 times, at a virus to cell ratio of 1:1 to 1:10,000, preferably about 1:100.

Preferably, the porcine cells comprise porcine kidney (PK) cells, for example PK TC/115 and PK 15 (ATCC CCL 33) cell lines.

Preferably, the cat cells comprise cat kidney cells, in particular Crandell cat kidney (CRFK) cells (ATCC CCL 94).

The live attenuated TGE virus can be propagated by cell culture in a suitable culture medium using mammalian cells The method comprises the steps of inoculating mammalian tissue culture cells with TGEV (the cells have normally been grown to an 80-100% confluent monolayer prior to inoculation), harvesting the cells, typically between 1 and 7 days, preferably between 2 and 5 days, after inoculation, and recovering the propagated virus from the harvested cells.

Suitable culture media for use in the above method include those known in the art. A preferred culture medium in which the TGE virus can be propagated is Eagle Minimum Essential Medium (MEM) with or without serum.

Cell and virus growth is maintained under standard conditions, normally at 33-39ºC, preferably at 35-38ºC.

Viral fluids are suitably inoculated into mammalian cells at a virus to cell ratio of 1:10 to 1:10,000, preferably 1:100. Preferably, the virus culture medium is added after a period of 1 to 2 hours.

The mammalian cells are suitably of pig or cat origin, for example pig kidney cells (PK) cells, in particular the PK 15 cell line (ATCC CCL 33), or cat kidney cells, in particular the Crandell cat kidney cells (CRFK) (ATCC CCL 94).

Preferably, the mammalian cells originate from cats, more preferably CRFK cells.

Viral fluids are preferably harvested approximately 3 days after inoculation by freezing and thawing or by enzymatic means. The harvested viral fluids can be stored frozen in the usual way.

Typically, viral fluids harvested as above contain at least 103.5 infectious virus units/ml.

If a live TGEV vaccine is required, the viral fluids can be used without further processing or they can be mixed with a suitable stabilizer, for example N-Z-amine gelatin, sucrose phosphate glutamate (SPG) or sucrose phosphate glutamate albumin (SPGA). Normally the vaccine is dried. Preferably the titre after drying is at least 10⁴ per dose.

If an inactivated (or "killed") vaccine is required, the harvested viral fluids can be inactivated by methods known in the art. Suitable methods include, for example, treatment with β-propiolactone formaldehyde or ethyleneimine. After an inactivation and incubation period, the viral fluids can be tested for complete inactivation by known methods, for example, by multiple passage on pig kidney cells.

Another preferred method for carrying out the inactivation step is to treat the harvested viral fluids with ascorbic acid and/or a salt thereof in the presence of oxygen and a heavy metal ion source. Viral inactivation methods of this type have been described in Chem. Abs. 107(5), 36455u (for bacteriophages), in Chem. Abs. 61(11), 13647b (for vaccinia virus) and in our pending EP-A-0 310 317 (for vaccine components).

Suitable forms of ascorbic acid include the alkali metal or alkaline earth metal salts, for example sodium, potassium or calcium salts, or ascorbic acid itself. A preferred salt is the sodium salt. A suitable ascorbate concentration for use in the inactivation step is in the range 1-10 mM, preferably 2.0 mM-6.0 mM, for example 2.5 mM.

Suitable sources of heavy metal ions include copper, iron and zinc salts and mercury-containing compounds. A preferred heavy metal ion is the copper (Cu²⁺) ion, the usual source of which is copper sulfate. Another preferred heavy metal ion is mercury, the usual source of which is organomercurial compounds, in particular the sodium salt of Ethylmercuric thiosalicylic acid (thimerosal). The heavy metal ion is present in a catalytic amount, a suitable concentration for use in the inactivation step being in the range of 0.01 mM to 0.1 mM, preferably 0.015 mM to 0.05 mM, for example 0.022 mM.

The presence of oxygen is essential and is usually provided by appropriate ventilation.

Normally the inactivation step is allowed to proceed between 1 and 54ºC, preferably at about 37ºC, until inactivation is found to be sufficient in a test. The inactivation time required is typically in the range of 10-100 hours, generally about 48-72 hours.

For the inactivation of certain viruses, for example parvovirus and rotavirus, further addition of ascorbic acid may be necessary to complete the inactivation process.

In addition, other agents known to inactivate viruses may optionally be added to complete the inactivation process. One such reagent is saponin, as described for example in Vet. Med. Nauki, 1980, 17(3), 45-55 (A. Motovski et al.), which may be added, for example, about 72 hours after addition of the ascorbic acid at a concentration of 0.5 mg/ml. Such a procedure has proven particularly useful in the inactivation of envelope viruses such as viruses of the herpes group, including PRV and IBR.

The progress of the inactivation process can occasionally be monitored by measuring the viability of the virus at intervals using standard techniques, for example, the indirect fluorescent antibody test to determine the residual virus titre.

To prepare the inactivated TGEV vaccine, the inactivation step is normally carried out by adding appropriate amounts of stock solutions of sodium ascorbate and copper sulfate to the stock virus and mixing the resulting solution in the presence of air at 37ºC for approximately 72 hours. After cooling to 4ºC, a test for inactivation can usually be done using a suitable cell culture, such as PK or CRFK.

The TGEV vaccine for use in the method of the invention may optionally contain an additional attenuated modified live virus or killed viruses such as canine distemper virus, canine parainfluenza virus, canine adenovirus (I and II), canine parvovirus, and may be combined with various inactivated bacterial vaccines such as Leptospira canicola-Icterusbacterin or canine Bordetellabacterin.

The inactivated TGEV vaccine composition of the invention may occasionally contain adjuvants such as aluminum hydroxide (for example 2- 25% by weight, preferably 5-25% by weight), saponin (for example 0.5-1.5 mg/dose) and Freund's incomplete adjuvant (typical water-in-oil emulsion).

For the prevention of canine coronavirus infection in dogs, the TGEV vaccine may be administered parenterally (subcutaneously or intramuscularly) at a dose of at least 1.9 log viral particles per dose, preferably at least 1,000 viral particles (3 log viral particles) per dose. Advantageously, the vaccine is administered at a dose of at least 4 log viral particles per dose.

The following examples illustrate the invention.

Examples

The following materials and methods are used in the examples described below.

A. TGE virus Preparation A.1

The Purdue stock TGE virus (ATCC VR-763) was passaged four times on pig kidney cells to produce an original stock virus (designated PK0809). The experimental vaccines were derived from the original stock virus after 5 additional passages in the pig kidney cells.

Preparation A.2

The original stock virus from preparation 1 was passaged three times in pig kidney cells and twice in Crandell cat kidney cells (ATCC CCL 94).

B. Cell culture Preparation B.1

The original stock virus was propagated at 35-38ºC on the porcine kidney cell line PK 15 (ATCC CCL 33) in Eagle MEM with not more than 10% bovine serum.

Preparation B.2

The virus from preparation A.2 was propagated at 35-38ºC on the Crandell cat kidney cell line (ATCC CCL 94) in Eagle MEM with not more than 10% bovine serum.

C. Virus growth Preparation C.1

Two small roller bottles containing confluent monolayers of PK0809 were inoculated with 1 ml of undiluted original TGE stock virus. After a 1-2 hour adsorption period, virus growth medium (Eagle MEM with no more than 2% bovine serum) was added and virus was grown at 35-38ºC.

Preparation C.2

Two small roller bottles containing confluent monolayers of van Crandell cat kidney cells (ATCC CCL 94) were inoculated with 1 ml of undiluted TGE main strain virus. After an adsorption time of 1-2 hours, the virus growth medium (Eagle MEM containing less than 2% bovine serum) was added and the virus was grown at 35-38ºC.

example 1 Modified live TGEV vaccine

To a roller bottle of TGE virus prepared as described above in Preparation C.1, 17% N-Z-amine gelatin stabilizer is added. A freeze-thaw cycle is performed and the resulting material is dried in 1 ml volumes and kept at 4°C for as long as necessary. The titer after drying should be at least 10⁴ per dose.

Example 2 Modified live TGEV vaccine

A modified live TGEV vaccine was prepared in the same manner as in Example 1, except that the TGE virus prepared as in Preparation C.2 was used.

Example 3 Inactivated TGEV vaccines

A roller bottle of TGE virus prepared as described above in Preparation C.1 was frozen and thawed (titer samples taken) and inactivated as follows (the stock virus should have a pre-inactivation titer of at least 105.5 per dose):

The stock virus was placed in a 1-liter glass bottle and brought to 37ºC. A sodium ascorbate stock solution (1% w/v) and a copper sulfate stock solution (1.1% w/v) were then added to bring the final concentration of ascorbate to 5.05 mM and the final concentration of copper ions to 0.022 mM. The contents of the bottle were mixed with appropriate aeration for 72 hours at 37ºC and then cooled to 4ºC. An inactivation test was carried out in pig kidney cells.

After inactivation was established, the parent virus was cleaved and adjuvants were added to obtain two inactivated vaccines, as follows.

Vaccine A

One half of the inactivated stock virus is mixed with 10% aluminum hydroxide while stirring for 15-20 hours at 4ºC.

Vaccine B

The other half of the inactivated stock virus is mixed with adjuvants as follows:

Solution 1 consists of 5 ml of emulsifier mixed with 95 ml of Drakoil 6 VR. "Drakoil" is a trademark.

Solution 2 consists of 2.5 ml Tween 80 mixed with 47.5 ml killed TGE virus.

Solution 1 (21ml) is added to solution 2 (7ml) and mixed using a Perfektum emulsifying needle prior to inoculation.

Example 4 Dose titration of modified live TGEV vaccine and determination of CCV immunogenicity in dogs a) Experimental pattern

Four TGEV vaccine permutations were determined; two antigenic amounts were administered intramuscularly (i.m.) or subcutaneously (s.c.). Five dogs were assigned to each i.m. group and 4 dogs to each s.c. group. Five dogs were used as non-vaccinated controls.

Dogs in each vaccination group received 2 ml doses of a modified live TGEV vaccine administered i.m. or s.c. 4 weeks apart. Three weeks after the second vaccination, all dogs were challenged orally and intratonsillarly with CCV. Three parameters were determined to determine the efficacy of each vaccine permutation: serological response, rectal CCV excretion, and CCV infection of intestinal epithelial cells 5 days after challenge.

b)Materials and methods i) Animals

Twenty-three mongrel dogs, 16-20 weeks old, that had never been vaccinated against CCV were used. The dogs were susceptible to CCV as shown by CCV-IFA titers of < 1:2.

ii) Vaccine and vaccination

The original TGEV stock virus was passaged three times in pig kidney cells and twice in Crandell cat kidney (CRFK) cells as described in preparation A.2. The harvested virus material was kept at -70ºC prior to use.

iii) Serology

Antibody titres were determined at the time of vaccination, challenge and 5 days post-challenge using an indirect fluorescent antibody assay.

iv) Exposure

A field isolate designated CCV-6 and passaged once in CRFK was used. Dogs were deprived of food for 24 hours prior to challenge. Four milliliters of harvested virus material was administered to each dog in the following manner: 2 ml of virus material mixed with 1/4 cup of Alpo was administered orally. Dogs were then anesthetized with Pram Ace (Ayerst Laboratories INC NY, NY 10017) according to the package insert recommendations and 1 ml of CCV was then injected into each palatine tonsil. Dogs were monitored on the day of challenge and for 5 days following the subsequent challenge for prominent clinical signs and rectal virus shedding.

v) Recovery of rectal virus after exposure

Rectal swabs were collected daily and processed for virus isolation by passage in CRFK cells.

vi) Small bowel assessment after CCV exposure

Five days after CCV challenge, all dogs were necropsied and the entire small intestine was removed. Intestinal impressions on slides were made from regularly shaped intestinal segments. The presence of CCV antigen was determined using an indirect fluorescent antibody test. The impression slides were scored according to the percentage of specific CCV fluorescence observed.

Results

No clinical signs were observed in dogs following vaccination or exposure.

i)Titre of the virus vaccine

The antigen amounts used in this study were 103.3 and 102.3 TCID₅�0 /dose.

ii) Serological response

The serological results are summarized in Table 1. Table 1 Reciprocal geometric mean CCV antibody titers TGE vaccine Weeks after first vaccination 5 days after exposure Unvaccinated

All dogs had no measurable antibody titer at the time of vaccination. Unvaccinated controls remained below 1:10 throughout the course of the study, indicating the absence of external CCV infection.

Dogs in all four vaccination groups (103.3 im, 103.3 sc, 102.3 im, and 102.3 sc) developed similar geometrically mean antibody titers (GMAT) within 3 weeks after the first vaccination (1:24 to 1:46), indicating a primary response to virus replication. Three weeks after the second vaccination, a dose-dependent response was demonstrated among dogs in the 103.3 im and sc groups (GMAT 1:139 and 1:113) and the 102.3 im and sc groups (GMAT 1:53 and 1:24). All vaccinated dogs demonstrated an anamnestic response to CCV challenge; responses among dogs in the 103.3 im and sc groups were 3-fold and responses among dogs in the 102.3 im and sc groups were nine- and eight-fold, respectively. The serological results show that the canine immune system stimulated by the heterologous live TGEV vaccine responded anamnestic to exposure to the homologous virulent CCV.

Unvaccinated dogs showed no measurable antibody response 5 days after exposure.

iii) Post-exposure CCV determinations

Duplicate titrations of CCV at challenge showed that dogs were exposed to 105.4 TCID₅₀/mL (106.0 TCID₅₀/dose).

Isolations of CCV virus were monitored for five days after challenge. All dogs were free of CCV prior to challenge. Inclusions of CCV virus within the CRFK cytoplasm were observed from all unvaccinated controls (100% recovery) during the 5-day period. Inclusions of CCV virus were not observed among vaccinated dogs, indicating that no virus was enriched. Passaging of the material did not enhance virus enrichment.

Table 2 summarizes the mean specific CCV intestinal fluorescence among the groups and unvaccinated controls. Table 2 Average CCV score (%) in the intestine by immunofluorescence Intestinal print number (duodenum to ileum) Group Control Total

Specific CCV fluorescence in the form of bright apple-green fluorescence was observed along the entire length of the small intestine. All dogs showed some degree of CCV infection. A significant decrease in intestinal fluorescence compared to controls was observed among all vaccinated dogs.

The mean overall score between vaccination groups 5 days after CCV challenge was similar (30.6, 56.3, 46.2, 42.5) and the mean overall score of the unvaccinated control dogs was significantly higher (327) than that of the vaccine groups.

d) Conclusion

Dogs in all vaccine groups developed similar antibody responses 3 weeks after the first vaccination. A dose-dependent antibody response was observed among dogs in the 103.3 and 102.3 vaccine groups 3 weeks after the second vaccination.

After CCV challenge, virus was recovered from all unvaccinated individuals and CCV intestinal infection appeared active and widespread. However, vaccinated dogs in all vaccine groups mounted an active cellular immune response without rectal virus shedding and were apparently protected from CCV challenge by TGE vaccine-elicited antibodies or cell-mediated immunity.

Claims (6)

1. Use of the infectious gastroenteritis virus from pigs for the manufacture of a vaccine for the prevention of canine coronavirus infection in dogs. 2. Use according to claim 1, wherein the vaccine is inactivated. 3. Use according to claim 1 or 2, wherein the vaccine is prepared from the Purdue strain TGE virus (ATCC No. VR-763). 4. Use according to any one of claims 1 to 3, wherein the vaccine additionally comprises an attenuated modified live virus or inactivated virus selected from canine distemper virus, canine parainfluenza virus, canine adenovirus (I and II), canine parvovirus and canine coronavirus, or is combined with an inactivated bacterial vaccine selected from Leptospira canicola-Icterusbacterin and canine Bordetellabacterin. 5. A process for the preparation of an inactivated vaccine prepared from infectious porcine gastroenteritis virus (a TGEV vaccine), comprising: (a) introducing a TGE virus isolate into pig and/or cat cells at a virus to cell ratio of 1:1 to 1:10,000 to yield a live attenuated TGE virus; (b) propagation of the live TGE virus by cell culture in mammalian cells; (c) harvesting the viral fluids so obtained; and (d) inactivating the viral fluids with ascorbic acid and/or a salt thereof in the presence of oxygen and a heavy metal ion source. 6. Use according to claim 2, further characterized in that the inactivated vaccine was prepared using the method according to claim 5.